Human DNA Topoisomerase Research Articles

Virtual screening and multilevel precision-based prioritisation of natural inhibitors targeting the ATPase domain of human DNA topoisomerase II alpha

Human DNA topoisomerase II alpha (hTopIIα) is a classic chemotherapeutic drug target. The existing hTopIIα poisons cause numerous side effects such as the development of cardiotoxicity, secondary malignancies, and multidrug resistance. The use of catalytic inhibitors targeting the ATP-binding cavity of the enzyme is considered a safer alternative due to the less deleterious mechanism of action. Hence, in this study, we carried out high throughput structure-based virtual screening of the NPASS natural product database against the ATPase domain of hTopIIα and identified the five best ligand hits. This was followed by comprehensive validation through molecular dynamics simulations, binding free energy calculation and ADMET analysis. On stringent multilevel prioritization, we identified promising natural product catalytic inhibitors that showed high binding affinity and stability within the ligand-binding cavity and may serve as ideal hits for anticancer drug development. Communicated by Ramaswamy H. Sarma

Phenotypic Discovery of Thiocarbohydrazone with Anticancer Properties and Catalytic Inhibition of Human DNA Topoisomerase IIα.

Phenotypic screening of α-substituted thiocarbohydrazones revealed promising activity of 1,5-bis(salicylidene)thiocarbohydrazide against leukemia and breast cancer cells. Supplementary cell-based studies indicated an impairment of DNA replication via the ROS-independent pathway. The structural similarity of α-substituted thiocarbohydrazone to previously published thiosemicarbazone catalytic inhibitors targeting the ATP-binding site of human DNA topoisomerase IIα prompted us to investigate the inhibition activity on this target. Thiocarbohydrazone acted as a catalytic inhibitor and did not intercalate the DNA molecule, which validated their engagement with this cancer target. A comprehensive computational assessment of molecular recognition for a selected thiosemicarbazone and thiocarbohydrazone provided useful information for further optimization of this discovered lead compound for chemotherapeutic anticancer drug discovery.

Binding profile of a mixed-ligand silver(I) complex with DNA and Topoisomerase I

A new mixed-ligand Ag(I) complex, [Ag(daf)(phen)]NO3 (daf = 4,5-diazafluoren-9-one and dian = N-(4,5-diazafluoren-9-ylidene)aniline), was synthesized. The elemental analysis, FTIR, 1HNMR, UV–Vis spectroscopy, cyclic voltammetry, and DFT (Density Functional Theory) geometry optimization method were applied in order to predict the Ag(I) complex structure which concluded to a distorted tetrahedral N4 coordination around the Ag(I) center. A detailed in silico analysis of the bioaffinity of the complex to DNA and human DNA-Topoisomerase I was conducted using molecular docking simulations and ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) techniques. In this overall scenario, the results suggest the dominance of π-π stacking interactions of the heteroaromatic ligands in the intercalating pocket of DNA and the active site of the enzyme and the rational correlation between being a good intercalator and a potent Topoisomerase I inhibitor. In vitro DNA-binding experiments by spectrophotometric, spectrofluorometric, Voltammetric, and viscometric techniques at physiological pH also confirmed the computational results. The complex inhibited MCF-7 cell growth in a dose-dependent manner while being nontoxic on HUVEC normal cells.

Molecular Docking and ADMET Study of Spice‐Derived Potential Phytochemicals against Human DNA Topoisomerase III Alpha

AbstractSmall phytochemicals play an important role in therapeutics hence potential molecules in different spices are screened using Lipinski's and Veber's rule. Pharmacodynamics studies are carried out against the cancer target, DNA topoisomerase III alpha. In silico ADMET (absorption, distribution, metabolism, excretion, toxicity) studies are done with these compounds. Lipinski's and Veber's rule indicates that all 10 small phytochemicals have the potential to behave as oral drug‐like molecules. A molecular docking study shows that shogoal, demethoxycurcumin, capsaicin, ellagic acid, 6‐paradol, 6‐gingerol, carnosic acid, and curcumin bind strongly with the cancer target: human DNA topoisomerase‐III alpha. In silico absorption, a study reveals that only cinnamic acid, allicin, alpha turmerone, cinnamyl acetate, and carnosic acid have more than 96% human intestinal absorption, but only alpha turmerone can cross both the blood–brain barrier and central nervous system. In silico phase‐I, the metabolism study shows 6‐paradol and carnosic acid as substrates for cytochrome 450 enzyme (CYP450 3A4) and curcumin and dillapiol as inhibitors for CYP 450 2D6. Overall the study concludes that small phytochemicals present in different spices have the potential to be proposed as oral anti‐cancer drugs against cancer targets DNA topoisomerase III alpha.

ATP-competitive inhibitors of human DNA topoisomerase IIα with improved antiproliferative activity based on N-phenylpyrrolamide scaffold

ATP-competitive inhibitors of human DNA topoisomerase II show potential for becoming the successors of topoisomerase II poisons, the clinically successful anticancer drugs. Based on our recent screening hits, we designed, synthesized and biologically evaluated new, improved series of N-phenylpyrrolamide DNA topoisomerase II inhibitors. Six structural classes were prepared to systematically explore the chemical space of N-phenylpyrrolamide based inhibitors. The most potent inhibitor, 47d, had an IC50 value of 0.67 μM against DNA topoisomerase IIα. Compound 53b showed exceptional activity on cancer cell lines with IC50 values of 130 nM against HepG2 and 140 nM against MCF-7 cancer cell lines. The reported compounds have no structurally similarity to published structures, they are metabolically stable, have reasonable solubility and thus can serve as promising leads in the development of anticancer ATP-competitive inhibitors of human DNA topoisomerase IIα.

Disease-associated H58Y mutation affects the nuclear dynamics of human DNA topoisomerase IIβ

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics.

Identification of the first-in-class dual inhibitors of human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1) with strong anticancer properties

Molecular docking of a large set of thiosemicarbazide-based ligands resulted in obtaining compounds that inhibited both human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase-1 (IDO1). To the best of our knowledge, these compounds are the first dual inhibitors targeting these two enzymes. As both of them participate in the anticancer response, the effect of the compounds on a panel of cancer cell lines was examined. Among the cell lines tested, lung cancer (A549) and melanoma (A375) cells were the most sensitive to compounds 1 (IC50=0.23 µg/ml), 2 (IC50=0.83 µg/ml) and 3 (IC50=0.25 µg/ml). The observed activity was even 90-fold higher than that of etoposide, with selectivity index values reaching 125. In-silico simulations showed that contact between 1-3 and human DNA topoisomerase II was maintained through aromatic moieties located at limiting edges of ligand molecules and intensive interactions of the thiosemicarbazide core with the DNA fragments present in the catalytic site of the enzyme.

Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B.

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein-DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.

A Computational QSAR, Molecular Docking and In Vitro Cytotoxicity Study of Novel Thiouracil-Based Drugs with Anticancer Activity against Human-DNA Topoisomerase II.

Computational chemistry, molecular docking, and drug design approaches, combined with the biochemical evaluation of the antitumor activity of selected derivatives of the thiouracil-based dihydroindeno pyrido pyrimidines against topoisomerase I and II. The IC50 of other cell lines including the normal human lung cell line W138, lung cancer cell line, A549, breast cancer cell line, MCF-7, cervical cancer, HeLa, and liver cancer cell line HepG2 was evaluated using biochemical methods. The global reactivity descriptors and physicochemical parameters were computed, showing good agreement with the Lipinski and Veber’s rules of the drug criteria. The molecular docking study of the ligands with the topoisomerase protein provides the binding sites, binding energies, and deactivation constant for the inhibition pocket. Various biochemical methods were used to evaluate the IC50 of the cell lines. The QSAR model was developed for colorectal cell line HCT as a case study. Four QSAR statistical models were predicted between the IC50 of the colorectal cell line HCT to correlate the anticancer activity and the computed physicochemical and quantum chemical global reactivity descriptors. The predictive power of the models indicates a good correlation between the observed and the predicted activity.

Discovery of 1,2-diphenylethene derivatives as human DNA topoisomerase II catalytic inhibitors and antitumor agents

Human DNA topoisomerase II (TopoII) is highly correlated with cell proliferation, and involved in tumor biogenesis and development. The classic chemotherapeutic agents etoposide (VP-16) and adriamycin (ADR) targeting TopoII are wildly used in clinical applications. Herein, fifty-eight pinosylvin (1,2-diphenylethene) derivatives as TopoII inhibitors were designed and synthesized through three generations of structural optimizations on the basis of the structure of the initial hit A1 from in-house chemical library. The most potent compound F2 showed high in vitro inhibitory efficacy against TopoII (IC50 α 3.8, β 10.1 μM), compared to that of VP-16 (IC50 α 110.0, β 36.1 μM) for pBR322 DNA relaxation with no evident TopoII poisons in DNA cleavage assay. Meanwhile, F2 exhibited strong antitumor activities against human cancer cell lines HeLa, HCT-116, PC-3, MDA-MB-231, HepG2 and A549 (IC50 0.1–0.3 μM), compared to that of VP-16 (IC50 1.5–15.1 μM). F2 showed less cytotoxicity against normal murine cell line CCL-226 (IC50 > 50 μM) than that of VP-16 (IC50 20.8 μM). The selectivity index of F2 and VP-16 are larger than 52.1 and 1.3–26.2 in cell lines, respectively. Additionally, F2 exhibited potent potency in apoptosis induction and cell cycle arrest in HepG2 cells. These results provide a promising strategy and good starting point for the development of potent TopoII inhibitors as antitumor agents.

Catalytic Mechanism of ATP Hydrolysis in the ATPase Domain of Human DNA Topoisomerase IIα.

Human DNA topoisomerase IIα is a biological nanomachine that regulates the topological changes of the DNA molecule and is considered a prime target for anticancer drugs. Despite intensive research, many atomic details about its mechanism of action remain unknown. We investigated the ATPase domain, a segment of the human DNA topoisomerase IIα, using all-atom molecular simulations, multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, and a point mutation study. The results suggested that the binding of ATP affects the overall dynamics of the ATPase dimer. Reaction modeling revealed that ATP hydrolysis favors the dissociative substrate-assisted reaction mechanism with the catalytic Glu87 serving to properly position and polarize the lytic water molecule. The point mutation study complemented our computational results, demonstrating that Lys378, part of the important QTK loop, acts as a stabilizing residue. The work aims to pave the way to a deeper understanding of these important molecular motors and to advance the development of new therapeutics.

Multiple gene copy number increases total protein expression and enzyme activity of DNA topoisomerase I in Pichia pastoris

The present study aimed to employ an <em>in vivo</em> strategy for the establishment of multi‑copy gene constructs of human DNA topoisomerase I (hTopI) using the pPIC3.5K vector in <em>Pichia</em>. The clones with multi‑copy inserts (His⁺ transformants) that were able to survive in the highest concentration of Geneticin^® were found to express the highest expression level of total protein and exhibited the highest target enzyme activity. The highest level of total protein found was 1.76 mg/ml in GS115‑pPIC3.5K‑hTop1, which was resistant to 1.00 mg/ml Geneticin at 48 h of incubation. The highest enzyme activity of hTopI was also observed in the culture expressed by GS115‑pPIC3.5K‑hTopI, which was resistant to 1.00 mg/ml Geneticin^® (19.7x104 Ul/OD₆₀₀). On the whole, the present study provides information regarding the production of target protein from recombinant <em>Pichia</em> using only a shaker flask system, which can be further developed as an in‑house resource for screening potential anticancer agents.

Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα.

Simple SummaryDNA topoisomerase IIα (170 kDa, TOP2α/170) resolves nucleic acid topological entanglements by generating transient double-strand DNA breaks. TOP2α inhibitors/poisons stabilize TOP2α-DNA covalent complexes resulting in persistent DNA damage and are frequently utilized to treat a variety of cancers. Acquired resistance to these chemotherapeutic agents is often associated with decreased TOP2α/170 expression levels. Studies have demonstrated that a reduction in TOP2α/170 results from a type of alternative polyadenylation designated intronic polyadenylation (IPA). As a consequence of IPA, variant TOP2α mRNA transcripts have been characterized that have resulted in the translation of C-terminal truncated TOP2α isoforms with altered biological activities. In this paper, an example is discussed where circumvention of acquired TOP2α-mediated drug resistance was achieved by utilizing CRISPR/Cas9 specific gene editing of an exon/intron boundary through homology directed repair (HDR) to reduce TOP2α IPA. These results illustrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant IPA.Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.

A comprehensive structural analysis of the ATPase domain of human DNA topoisomerase II beta bound to AMPPNP, ADP, and the bisdioxopiperazine, ICRF193

SummaryHuman topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.

POSSIBLE CARDIOPROTECTIVE MECHANISM OF ACTION OF DEXRAZOXANE, AND PROBABLE HUMAN TOPOISOMERASE IIβ INHIBITORS: AN IN SILICO ANALYSIS

Objective: The aim of this study was to determine which metabolite plays a role in the cardioprotective effect of dexrazoxane, and also to identify alternative compounds to dexrazoxane since clinical use of dexrazoxane is limited. For this purpose, the interactions of dexrazoxane and its three metabolites (B, C, and ADR-925), as well as the compounds, which reported to be inhibitors for topoisomerase VI (prototype of human DNA topoisomerase II beta), with human DNA topoisomerase II beta were investigated by molecular docking. Afterwards, the theoretical ADMET properties of all these compounds were determined Material and Method: The molecular structures were optimized by Gaussview 05 and Gaussian 03 package programs. AutoDock 4.2 software was used for molecular docking studies and the docking complexes were analyzed in 2D and 3D using the Discovery Studio Client 4.1 program. The pkCSM online program was used to calculate the theoretical ADMET parameters. Result and Discussion: As a result of molecular docking studies, it was determined that the B metabolite of dexrazoxane has a higher binding potential to human DNA topoisomerase II beta compared to both dexrazoxane and its other metabolites. The binding potentials of other compounds reported in the literature to human DNA topoisomerase II beta were radicicol>quinacrine>purpurin>9-Aminoacridine>hexylresorcinol, respectively. The results showed that the B metabolite of dexrazoxane plays an important role in the cardioprotective mechanism of action of dexrazoxane against anthracycline cardiotoxicity. In addition, it has been determined that other compounds, except purpurin, have the potential to cause toxicity.

Discovery of 1,2-Diphenylethene Derivatives as Human DNA Topoisomerase Ii Catalytic Inhibitors and Antitumor Agents

Discovery of 1,2-Diphenylethene Derivatives as Human DNA Topoisomerase Ii Catalytic Inhibitors and Antitumor Agents

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21

DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

Preparation, characterization and evaluation of [125I]-pirarubicin: A new therapeutic agent for urinary bladder cancer with potential for use as theranostic agent

Improving urinary bladder cancer diagnosis, follow-up, and therapy tools to overcome existing limitations and increase survival rates is a highly desirable goal. In the current investigation, pirarubicin, a new generation antineoplastic anthracycline, was labeled with [125I] via an electrophilic substitution reaction. The reaction parameters were studied to optimize the iodination process. The labeled compound showed high radiochemical yield (98.5 ± 2.1%) and consistently remained above 90% for more than 20 h at room temperature and in the presence of serum at 37 °C. The binding of [125I]-pirarubicin to its target DNA-human topoisomerase II complex was assessed in-silico. The in-vitro tracer uptake by cancer cells was high and reached saturation (88.4 ± 2.3%) after 3 h with nuclei to cells ratio of 40 ± 1.2%. The labeled compound antiproliferative effect was much stronger than the unlabelled pirarubicin, as cleared by the growth inhibition test. Radiotoxicity improved cancer cells drug cytotoxicity. The in-vivo evaluation results showed that the [125I]-pirarubicin tends to preferentially accumulate in urinary bladder cancerous tissues.

Exploration of the Role of the C-Terminal Domain of Human DNA Topoisomerase IIα in Catalytic Activity.

Human topoisomerase IIα (TOP2A) is a vital nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. TOP2A is a homodimer with a symmetrical, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but is involved in enzyme regulation and is predicted to be intrinsically disordered. In addition, it appears to be a major region of post-translational modification and includes several Ser and Thr residues, many of which have not been studied for biochemical effects. Therefore, we generated a series of human TOP2A mutants where we changed specific Ser and Thr residues in the C-terminal domain to Ala, Gly, or Ile residues. We designed, purified, and examined 11 mutant TOP2A enzymes. The amino acid changes were made between positions 1272 and 1525 with 1–7 residues changed per mutant. Several mutants displayed increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272–1279, 1324–1343, 1351–1365, and 1374–1377 produced 2–3 times more DNA cleavage in the presence of etoposide than wild-type TOP2A. Further, several mutants displayed changes in relaxation and/or decatenation activity. Together, these results support previous findings that the C-terminal domain of TOP2A influences catalytic activity and interacts with the substrate DNA. Furthermore, we hypothesize that it may be possible to regulate the enzyme by targeting positions in the C-terminal domain. Because the C-terminal domain differs between the two human TOP2 isoforms, this strategy may provide a means for selectively targeting TOP2A for therapeutic inhibition. Additional studies are warranted to explore these results in more detail.